Immumount

For top-down images, scaffolds were washed twice in PBS, fixed in 10% NBF for 30 min, washed twice in PBS for 5 min each and incubated in block solution for 2 h at room temperature. Primary antibodies were applied in block solution and incubated overnight at 4 °C. After three 5-minute washes in PBS, species-specific secondary antibodies were applied at 1:200 in block solution at room temperature for 1 h. Scaffolds were washed twice in PBS and DAPI (5 mg·mL⁻¹ stock, 1:10 000 in PBS; Life Technologies) was applied. Samples were washed in PBS before imaging, either in situ or following removal from wells and cover-slipping using ImmuMount (GeneTex, Irvine, USA), using a Zeiss 700 confocal microscope (Zeiss).
To assess cell viability on the surface of scaffolds, a live/dead cell viability assay was used (ThermoFisher Scientific). Calcein AM (3.4 µL·mL⁻¹) and ethidium homodimer-1 (4 µL·mL⁻¹) were mixed with medium and added to scaffolds following cell adhesion. The scaffolds were incubated for 45 min and mounted on glass slides for top-down, whole-mount imaging on a Zeiss 700 confocal microscope (Zeiss).

Slides were dewaxed for immunofluorescence using an automated process (DRS-601, Sakura) and then washed in distilled water and blocked with PBS containing 10% FBS (block solution) for 2 h. Primary antibodies were applied in block solution and incubated at 4 °C overnight. Following three PBS washes, species-specific secondary antibodies (AlexaFluor, ThermoFisher Scientific) were applied in block solution at a concentration of 1:200 for 1 h at room temperature. Slides were washed a further two times in PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI) (5 mg·mL⁻¹ stock, 1:10 000 in PBS; Life Technologies) for 10 min. Following a final PBS washing step, slides were cover-slipped using ImmuMount (GeneTex, Irvine, USA), sealed with nail varnish and imaged on a Zeiss 700 confocal microscope (Zeiss, Oberkochen, Germany).